Method for diagnosis of infectious mononucleosis

ABSTRACT

A method for preparing a storageable absorbent for diagnosis of infectious mononucleosis from blood serum is disclosed. The absorbent is prepared by treating mammal red blood cells with a proteolytic enzyme or neuraminidase to destroy the mononucleosis receptors and the red blood cells are hemolyzed to make a cellfree stroma suspension which does not interfere with the agglutination step of the diagnosis, when mammal blood is added to the mixture of blood serum to be tested and the absorbent on a slide.

United States Patent [1 1 Leikola 1 Feb.4, 1975 METHOD FOR DIAGNOSIS OF INFECTIOUS MONONUCLEOSIS [75] Inventor: E. Juhani Leikola, Helsinki, Finland 21 Appl. No.2 243,421

[30] Foreign Application Priority Data Apr. 14, 1971 Finland 1032 [52] U.S. Cl 424/12, 195/1035, 424/8 [51] Int. CL... G0ln 31/02, G0ln 33/16, C12k l/OO [58] Field of Search ..424/8,11, 12, 13;

[56] References Cited OTHER PUBLICATIONS Williams and Chase, Methods in lmmuno. and Immunochem Academic Press, NY. Vol. 111, 1971 pp 370-374 Springer, JLab & Clin Med, Vol. 65, 1965 pp 617-627 Tomcsik, PSEBM, Vol. 69, 1948 pp 562-565 Tomcsik, Path. and Microbio. Vol. 23, 1960 pp 172-183 Davidsohn, AJCP, Vol. 41, 1964 pp 115-125 Chem Abs, Vol. 65, 1966 pp 7,799-7,800

Primary Examiner-Albert T. Meyers Assistant ExaminerA. Fagelson Attorney, Agent, or Firm-Biebel, French & Bugg [57] ABSTRACT A method for preparing a storageable absorbent for diagnosis of infectious mononucleosis from blood serum is disclosed. The absorbent is prepared by treating mammal red blood cells with a ptoteolytic enzyme or neuraminidase to destroy the mononucleosis receptors and the red blood cells are hemolyzed to make a cell-free stroma suspension which does not interfere with the agglutination step of the diagnosis, when mammal blood is added to the mixture of blood serum to be tested and the absorbent on a slide.

6 Claims, No Drawings METHOD FOR DIAGNOSIS OF INFECTIOUS MONONUCLEOSIS BACKGROUND OF THE INVENTION 1. Field of the Invention The invention relates to the field of preparing a stable absorbent for diagnosis of infectious mononucleosis from blood serum and a method for carrying out the diagnosis.

2. Description of the Prior Art Infectious mononucleosis is a rather common viral disease occuring mainly in adolescents. The diagnosis of this disease is based mainly on the demonstration of an antibody in the blood serum of the patient which agglutinates sheep or horse red blood cells (RBC). In certain circumstances the antibody can also agglutinate beef RBC. However, since normal personssometimes have antibodies with similar properties, it is necessary to absorb these natural antibodies before it can be concluded that the agglutination reaction is due only to the specific mononucleosis antibodies.

The presence of the specific mononucleosis antibody can be demonstrated by making serial dilutions of the patients serum in test tubes, or by simple slide tests. In the original method of serial dilutions (Davidsohn, I., Stern, K., and Kashiwagi, C., Amer. J. Clin. Path. 21. l 101 1951) ground guinea pig kidney tissue was added to the serum to be tested to absorb the unspecific, natural antibodies. Guinea pig kidney tissue, however, is not ideal for this purpose, since it is able to absorb only part of the natural antibodies, and, on the other hand, it sometimes removes some part of the specific mononucleosis antibody. Since the absorption with guinea pig kidney alone cannot render the test sufficiently specific, this method includes another, simultaneous absorption. This confirming absorption is based on the fact that beef RBC have a reversed effect on the patients serum as compared with guinea pig kidney tissue, i.e., they absorb the specific mononucleosis antibody but leave the natural antibodies intact. After these two simultaneous absorptions have been made on the serum to be tested, two serial dilutions are made of the serum in test tubes, and a certain concentration of sheep RBC suspension is added into the tubes (horse RBC can be used as well). If the cells agglutinate after absorption of the serum with guinea pig kidney, but not after the confirming absorption with beef RBC, the reaction is concluded to be due to the specific mononucleosis antibody, and, hence, the diagnosis of infectious mononucleosis can be established. In the reversed case, i.e., there is no agglutination after absorption with guinea pig kidney but some degree of agglutination remains after the confirming absorption, this is due to the natural, unspecific antibodies.

This tube dilution method has recently been simplified by developing a slide test (Lee, C.L., Davidsohn, I., and Panczyszyn, 0., Amer. J. Clin. Path. 49: 12, I968 since the tube test with dilutions and centrifugations is rather laborious to perform. The principle of the slide test is similar to the tube test, but a slide is used instead of the tubes, and the patients serum is not diluted. In practice, two drops ofserum are put on a microscopic slide (or corresponding background). To the first drop, very finely ground guinea pig kidney is added, and to the second drop, beef RBC stromata are added. These'stromata are prepared by hemolyzing the ill beef RBC, and removing the hemoglobin. After a certain, short time, sheep (or, most frequently, horse) RBC are added to both drops. In positive cases. visible agglutination usually forms within 1-2 minutes. The criteria for interpretation of the test are the same as with the tube method.

Since the guinea pig kidney tissue is unsatisfactory in the absorption, and a confirmatory absorption is needed to make the test specific, a better absorbent has long been sought. One such absorbent was found in I955. This was prepared by destroying those parts of the sheep RBC membrane which carry the receptor for the mononucleosis antibody. but at the same time leaving the receptors for natural antibodies intact. This can be performed by a variety of proteolytic enzymes (best by papain) as well as by neuraminidase (Wollner, D., Zschr. Immunforsch, 1 I2: 290, I955; Wollmer, D., Azchr. Immunforsch. I13: 30l, I956; Davidsohn, I., Zschr. Lee, C.L., Amer. J. Clin. Path. 41: I15, l964; Springer, G. F. and Callahan, H. J., J. Lab. Clin. Med. 617, 1965). Thus, such enzyme-treated sheep RBC are excellent for absorption, since the removal of natural antibodies is completely without any effect on the mononucleosis antibody. This principle works very well in the tube dilution method. However, it is somewhat inconvenient since treated sheep RBC are unstable, as is papain. This requires new preparations of the absorbent for each test day. For this reason this theoretically very good test is not widely used routinely.

The present method is based on the same principle as described in the Description of the Prior Art above. However, a more advantageous slide modification has been developed for the test and the problem of stability has been solved. In addition to the greater specificity and sensitivity, it is also simplier than the present slide tests which are based on the principle of dual absorption. In this method, a large amount of sheep RBC are treated with enzyme. After the treatment, the RBC are carefully washed with physiological saline (0.9 weightpercent NaCl), and then hemolyzed. The hemoglobin is removed anda fine suspension is made of the resulting stromata. The cells are first treated with enzyme and only thereafter hemol'yzed, since the cell membrane is stret ched in the intact RBC and thus readily ac; cessible to the enzyme. The fine stro niasiispension is stable for at least several months especially if an appropriate preservative is added, and the suspension does not intefere with the final agglutination reaction, since the suspension does not contain any cells.

Since this slide test method is both specific and simple with good stability of the reagents, it can be used in circumstances where there is no convenient access to a serological laboratory. It also becomes less expensive than the methods with two absorbents.

SUMMARY OF THE INVENTION According to the invention there is prepared an absorbent which is storageable and suitable for slide tests by homolyzing the red blood cells before or after these have been treated with a proteolytic enzyme or neuraminidase in order todestroy the cells and prepare a fine stroma suspension which does not interfere with the slide test diagnosis.

DESCRIPTION OF THE PREFERRED EMBODIMENT More than two thousand sera from patients with infectious mononucleosis and normal, healthy persons have been tested by this method, and the great specificity and sensitivity has been proved.

The following reagents are required for the method: (a) the blood sereum to be tested, (b) the absorbent, (c) sheep blood.

The serum is removed from the whole blood by allowing the blood to coagulate. After separation of the serum from the clot, the serum is heated at 56C for 15 min to destroy the complement.

The absorbent is prepared in the following way, Sheep blood is taken into an anticoagulant medium to prevent the clotting of the blood. The blood cells are washed four times with 0.9 percent NaCl solution, centrifuging the cells between each washing. One part of washed sheep blood cells is mixed with four parts of 0.9 percent NaCl solution, and one part of papain solution is added (this is prepared by dissolving 1 gram of dry papain into 100 ml of0.l5M phosphate buffer, pH 5.4, and into this solution, 0.5M cystein is added to activate the enzyme). The mixture is incubated in 37C water bath until the mononucleosis receptors are destroyed (usually 60-120 min). After this treatment, the blood cells are washed several times with 0.9 mg digitonin in 1 ml of water). The RBC hemolyze, and the resulting stromata are washed at least 5 times by centrifuging the suspension at 12,000 rpm for min each time. The washed stromata are made into a suspension (usually l0- pecent) in 0.9 percent NaCl solution that is buffered to pH 7.2 with 0.02M phosphate buffer. As preservative, sodium azine is added in a ratio of l to 2,000.

The sheep blood is taken into an anticoagulant medium (e.g. Alsevers solution).

In practice, the test is performed as follows. One drop of the serum to be tested is put on a suitable slide (e.g.,

microscopic slide). One drop ofthe absorbent is added. and these two drops are thoroughly mixed using a glass or wooden applicator rod. After one minute of incubation at room temperature, a drop of sheep blood is added. This is again mixed, and the slide is manually slightly tilted each way to further mix the cells with the fluids. An agglutination will be seen after 1-2 min if the sample contains mononucleosis antibodies.

What is claimed is:

l. A method for diagnosis of infectious mononucleosis comprising forming an absorbent by treating sheep or horse red blood cells with a proteolytic enzyme or neuraminidaze in order to destroy the mononucleosis receptors, hemolyzing the red blood cells by adding a hemolyzing agent, separating the resulting stromata from the hemoglobin, and preparing a stroma suspension from the stromata and diluted saline which suspension is free from blood cells interfering with the diagnosis; and mixing blood serum to be tested, said absorbent and sheep or horse blood on a slide whereby if mononucleosis antibodies are present agglutination takes place.

2. The method of claim 1 wherein said sheep or horse blood mixed with said absorbent is in an anticoagulant medium.

3. The method ofclaim 1, wherein the red blood cells are washed with diluted saline after the enzyme treatment.

4. The method of claim 1, wherein an aqueous digitonin solution is used as the hemolyzing agent.

5. The method of claim 1, wherein a preservzitive is added to the stromata suspension.

6. The method of claim 5, wherein sodium azide is used as the preservative. 

1. A METHOD FOR DIAGNOSIS OF INFECTIOUS MONONUCLEOSIS COMPRISING FORMING AN ABSORBENT BY TREATING SHEEP OR HORSE RED BLOOD CELLS WITH A PROTEOLYTIC ENZYME OR NEURAMINIDAZE IN ORDER TO DESTROY THE MONONUCLEOSIS RECEPTORS, HEMOLYZING THE RED BLOOD CELLS BY ADDING A HEMOLYZING AGENT, SEPARATING THE RESULTING STROMATA FROM THE HEMOGLOBIN, AND PREPARING A STROMA SUSPENSION FROM THE STROMATA AND DILUTED SALINE WHICH SUSPENSION IS FREE FROM BLOOD CELLS INTERFERING WITH THE DIAGNOSIS; AND MIXING BLOOD SERUM TO BE TESTED, SAID ABSORBENT AND SHEEP OR HORSE BLOOD ON A SLIDE WHEREBY IF MONONUCLEOSIS ANTIBODIES ARE PRESENT AGGLUTINATION TAKES PLACE.
 2. The method of claim 1 wherein said sheep or horse blood mixed with said absorbent is in an anticoagulant medium.
 3. The method of claim 1, wherein the red blood cells are washed with diluted saline after the enzyme treatment.
 4. The method of claim 1, wherein an aqueous digitonin solution is used as the hemolyzing agent.
 5. The method of claim 1, wherein a preservative is added to the stromata suspension.
 6. The method of claim 5, wherein sodium azide is used as the preservative. 